The present invention relates to conjugated compounds consisting of heat shock proteins and polysaccharides or oligosaccharides, in particular those polysacccharides or oligosaccharides derived from the capsule of pathogenic microorganisms. Such compounds are capable of inducing the formation of anti-polysaccharide antibodies and are accordingly useful as vaccines for use in man and in animals.
Bacteria are the aetiological agents for a wide range of disease conditions.
Examples of such diseases include meningitis caused by Neisseria meningitidis and other infections caused by Haemophilus influenzae Type b (Hib) or Streptococcus (including Pneumococcus), typhoid fever caused by infection with Salmonella typhi, intestinal disease caused by non-typoidal Salmonella or Shigella bacteria.
It is known that protective immunity to capsular bacteria is mediated by antibodies to the capsular polysaccharides. It is also known that, in order to obtain sufficient stimulation of the immune system, it is necessary to conjugate capsular polysaccharides to carrier proteins (Robbins et al, J. Infect. Dis., 1990, 161,821-832).
In particular, there have been described in the literature conjugated compounds consisting of polysaccharides (for example Group C meningococcal polysaccharide (MenC), Hib and Group A meningococcal polysaccharide (MenA)) and proteins such as CRM-197 (a peptide derived from Corynebacterium diphtheriae), TD (Diphtheria toxoid) or TT (Tetanus toxoidxe2x80x94see Peeters et al. Inf.Immun., (October 1991), 3504-3510; Claesson et al., J. Pediatrics St Louis, 112(5), 695-702, (May 1988).
Some such vaccines are already used with good results in clinical practice. However, there exists the need to identify novel protein carriers which impart to the conjugates immunogenic properties better than those achieved with the carriers used hitherto.
The present invention relates to the use of heat shock proteins as a protein carrier to increase the immunogenic response of oligosaccharides and polysaccharides.
Heat shock proteins are known to contain a significant number of T epitopes and thus to stimulate the cellular immune system.
A conjugated compound of the heat shock protein of Mycobacterium bovis (65 kDa), as a carrier for a malarial epitope, has been described as inducing a marked immunity in animals pre-immunised with Bacillus Calmette-Guxc3xa9rin (BCG) without requiring adjuvants (Lussow et al Eur. J. Immunol., 1991, 21,2297-2302). It is however to be noted that the effects observed in Lussow et al relate to T cell dependent effects exhibited by peptides (which are well known to be T-cell dependent) conjugated to heat shock proteins.
More particularly, because the heat shock proteins are well conserved across bacteria of different strains and type, adventitious infection with bacteria, which is a continuous process, will ensure that the immune system remains sensitised to heat shock proteins, thus ensuring a good response to the conjugate compounds of the invention either at primary vaccination or on administration of a booster vaccination.
The present invention permits the use of bacterial capsular polysaccharides and oligosaccharides to be used without adjuvants (although adjuvants can be used).
Large numbers of children are given BCG vaccine (which will include bacterial heat shock proteins) to guard against tuberculosis and therefore a conjugate of the present invention containing a heat shock protein as the carrier would find a large number of subjects already pre-immunised with the carrier precisely as a result of the BCG vaccination which they have undergone.
Again, since the heat shock proteins are highly conserved even the population which has not been vaccinated with BCG can easily develop immunity (as a result of natural interaction with other bacteria) and can hence find itself in a state of being able to develop a good immune response following vaccination with a conjugate composed of a heat shock protein and a T cell-independent antigen (oligosaccharide or polysaccharide). Thus the carriers of the present invention uniquely exploit the high conservation of heat shock proteins across bacteria and T-cell memory to ensure high titre vaccination.
According to the present invention, there is provided a conjugate compound comprising at least one heat shock protein or portion thereof including at least one immunostimulatory domain and at least one oligosaccharide or polysaccharide.
The heat shock protein may be any heat shock protein capable of exhibiting an immunostimulatory effect in animals, preferably humans.
The heat shock proteins are highly conserved in bacteria, parasites and mammals. Any heat shock protein can be used in the conjugates of the present invention, provided it exhibits a positive immunostimulatory effect in the intended immunisation subject without significant deleterious effects. Specific, non-limiting examples include heat shock proteins from Helicobacter pylori, P. aeruginosa, C. trachomatis and M. leprae, especially the hsp60 group of heat shock proteins.
More particularly, three heat shock proteins are specifically exemplified herein, namely, M. bovis BCG GroEL-type 65 kDa hsp (hspR65), Recombinant M.tuberculosis DnaK-type 70 kDa hsp (hspR70) and a novel heat shock protein from H.pylori. 
The H. pylori heat shock protein (hsp) is a protein whose nucleotide and amino acid sequence is given in FIG. 3 and whose molecular weight is in the range of 54-62 kDa, preferably about 58-60 kDa. This hsp belongs to the group of Gram negative bacteria heat shock proteins, hsp60. In general, hsp are among the most conserved proteins in all living organisms, either prokaryotic and eukaroytic, animals and plants, and the conservation is spread along the whole sequence.
The conjugate may contain one or more heat shock proteins or immunostimulatory domains thereof. The heat shock proteins may the same or different. Preferably however, one heat shock protein or a portion containing one or more immunostimulatory domains is present.
As used herein, the term xe2x80x9cimmunostimulatory domainxe2x80x9d refers to a region of a heat shock protein amino acid sequence capable of enhancing the immune reaction of a subject mammal to a polysaccharide or oligosaccharide component of a conjugate compound including the domain.
An advantage of using only specific domains from complete heat shock proteins is that it is possible selectively not to include domains common to human heat shock proteins. For human vaccination this is advantageous as such regions will not affect the immunostimulatory effect of the heat shock protein as they will be recognized as xe2x80x9cselfxe2x80x9d. In addition any immunity that is stimulated against such xe2x80x9cselfxe2x80x9d regions might lead to autoimmunity.
Suitable domains of the hsp60 family of heat shock protein are identified in FIG. 2 by underlining of sequence of reduced homology with the human heat shock protein. Functional sub domains within the domains shown in FIG. 2 (SEQ. ID NO: 1); (SEQ. ID NO: 2); (SEQ. ID NO: 3); (SEQ. ID NO: 4); and (SEQ. ID NO: 5) may also be used, as can domain and sub domain combinations.
The skilled man can readily ascertain for a given heat shock protein which domains or epitopes are responsible for the immunostimulatory action and prepare modified heat shock protein containing only those domains or a sub set thereof.
The oligosaccharide or polysaccharide component of the conjugate compound may be the complete capsular polysaccharide or oligosaccharide of any pathogenic microorganism against which vaccination is indicated or a portion thereof capable of eliciting protective immunity. The oligosaccharide or polysaccharide may be from a single bacteria or from two or more bacteria.
Particular non-limiting examples of bacteria which may be targeted include: Haemophilus influenzae Type b (Hib), Streptococcus (including pneumococcus), Salmonella especially Salmonella typhi, intestinal disease caused by non-typoidal Salmonella or Shigella bacteria.
According to a particular embodiment of the invention, there have been prepared conjugates consisting of oligosaccharides of the Meningococci C (MenC) group and hsp.
According to a further particular embodiment of the present invention the hsp used for this purpose are hspR65 and hspR70.
In a second aspect of the invention, there is provided a process for producing conjugate compounds according to the present invention which comprises covalently bonding a heat shock protein or portion thereof including at least one immunostimulatory domain thereof to at least one oligosaccharide or polysaccharide.
The oligosaccharide or polysaccharide is preferably isolated from the bacterium to be targetted, but may be produced synthetically.
The heat shock protein may be isolated from its naturally occurring source or produced synthetically. Preferably the heat shock protein is produced by recombinant DNA technology using the techniques described in the general of the description herein.
Preferably the oligosacpharide or polysacoharide is modified prior to conjugation with the heat shock protein or portion thereof to provide reactive sites for conjugation. Suitably this involves introducing active functional groups, such as amino groups at the end groups of the oligosaccharide or polysaccharide. The thus modified oligosaccharide or polysaccharide may then be activated using a linking group, such as succinimide and conjugated to the heat shock protein or portion thereof.
In a third aspect of the invention, there is provided a conjugate compound according to the first aspect of the invention for use as a pharmaceutical, preferably as vaccine.
In a fourth aspect of the invention, there is provided the use of the conjugate compound according to the first aspect of the invention in the manufacture of a medicament for vaccination against bacterial infection.
In a fifth aspect of the invention, there is provided a method of vaccination comprising administering an immunologically effective amount of a conjugate compound according to the first aspect of the invention.
In a sixth aspect of the invention, there is provided a vaccine or therapeutic composition comprising one or more conjugate compounds according to the first aspect of the invention and a pharmaceutically acceptable carrier.
Preferably the composition is a vaccine composition and may include other excipients such as adjuvants, as necessary (see Section entitled xe2x80x9cVaccinesxe2x80x9d in the description below).
In a seventh aspect of the invention, there is provided a method for the preparation of a vaccine comprising bringing one or more conjugate compounds of the first aspect of the invention into association with a pharmaceutically acceptable carrier and optionally an adjuvant.